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4788 Afr. J. Agric. Res. solutions, due to concerns of expense, exposure risks fungicide residues and other health and environmental hazards. There is a vital need for alternative methods of control for coleus root rot. So far, effective and ecologically sound management practices have to be developed for managing this disease. Therefore, the objectives of the current study have been developed based on biological control strategy for this disease that is economically viable, environmentally safe durable and is an alternative to agrochemicals. Induced resistance has emerged as a potential tool in crop protection practices based on biological control. Induced resistance can be defined as the phenomenon by which plants exhibit increased levels of resistance to a broad spectrum of pathogens by the prior activation of genetically programmed defence pathways. The most extensively studied type of induced resistance is systemic acquired resistance (SAR) (Durrant and Dong, 2004). SAR is expressed locally and systemically after a localised infection by a necrotising pathogen or the application of some chemicals such as benzothiadiazole (BTH) (Conrath et al., 2001, 2006) and is characterised by the accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. The colonization of roots with selected plant growth-promoting rhizobacteria (PGPR) (De Vleesschauwer et al., 2006; Saravanakumar et al., 2007) can also lead to a type of systemic resistance, commonly denoted as induced systemic resistance (ISR). PGPR are a group of free-living saprophytic bacterial microorganisms that live in the plant rhizosphere and aggressively colonize the root system. They are studied as plant growth-promoters for increasing agricultural production and asbiocontrol agents against plant diseases (Chen et al., 2000; Saravanakumar et al., 2007). More specifically, the soil-borne fluorescent pseudomonads have received particular attention because of their catabolic versatility, excellent root colonising ability and their capacity to produce a wide range of enzymes and metabolites that favour the plant to withstand varied biotic and abiotic stress conditions (Radjacommare et al., 2004; Saravanakumar and Samiyappan, 2007). On the other hand, microorganisms as biological control agents often exhibit inconsistent performance in practical agriculture, resulting in limited commercial use of biocontrol approaches for the suppression of plant pathogens. For this reason, we evaluated fluorescent pseudomonads strains for their ability to promote Coleus plant growth and their effectiveness against root rot disease under glasshouse and field conditions. MATERIALS AND METHODS Isolation and collection of PGPR strains Rhizosphere soil from coleus fields was collected from districts of Salem and Coimbatore. The Fluorescent pseudomonad strains were isolated from the collected soil samples by using the serial dilution technique in Kings’ B medium (Peptone 20 g; MgSO 4 1.5 g; K 2HPO 4 1.5 g; Glycerol 10 ml; Agar 20; Distilled water 1l) . The strains of Pseudomonas sp. were identified according to the description given in ‘Bergey’s manual for systematic bacteriology’ (Krieg and Holt, 1984). These isolates were designated as follows and were maintained at -80°C with 50% glycerol. Twenty four elite strains of Pseudomonas sp. viz., PAO-1, PFKO-1, FP 7, PRA-1, MDU-1, PCN-3, PSKK-7, PPOL-3, 30-48-R1, PB-2-79, COGP-20, COGP- 30, COTP-20, COPP-36, COKP-46, TDK-1, AH-1, Pf1, PFNL-1, PRA-4, PSA-2, PSA-1, PPOL-1 and MDU-2 were obtained from the Culture Collection, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore and used for the study. Antifungal activity of fluorescent pseudomonads Fluorescent pseudomonad strains were tested for their antagonistic activity against Coleus root rot pathogen, M. phaseolina. A mycelial disc (8-mm diameter) from seven days old M. phaseolina was placed on one side of the Petri dishes containing an equal amount of KB and PDA medium. Pseudomonas cultures were streaked on the medium exactly opposites to the mycelial disc. The plates were incubated at room temperature (28 ± 2°C) for 6 days. Efficiency of the antagonistic organisms against the root rot pathogen was rated based on the inhibition zone observed (Rabindran and Vidyasekaran, 1996). The percent inhibition of growth of the test pathogen was calculated by using the formula where, I = Percent inhibition; C = Growth (cm) in control; T = Growth (cm) in treatment. The effective isolates from dual plate technique were tested for their actual mode of action. Mechanism of action of PGPR on plant pathogenic fungi Indole acetic acid (IAA) production The production of indole acetic acid (IAA) by Pseudomonas isolates was determined by a modified method of Gupta (1999). Bacterial isolates were grown in nutrient broth supplemented with tryptophane (5 mg/ml). Five millilitres of each bacterial culture was centrifuged at 2816 g for 15 min. The supernatant was collected and filtered through filter paper. Two millilitres of supernatant was mixed with two drops of O-phosphoric acid and 4 ml of freshly prepared Solawaski’s reagent (50 ml of 35% perchloric acid, 1 ml 0.5% FeCl 3). IAA development caused the production of a pink colour, which was measured by reading its absorbance at 530 nm. The level of IAA produced was estimated using a standard IAA graph. Production of siderophores Production of siderophores was estimated qualitatively on Chrom- Azurol S agar medium adjusted to pH 6.8 with NaOH (Schwyn and Neilands, 1987). An actively growing culture of each Pseudomonas isolate was spotted onto the Chrom-Azurol S plates and incubated for 24 to 48 h at 32°C. The appearance of a bright zone with yellowish fluorescent colour in the dark blue medium was the indication of production of siderophore. Production of siderophore was scored as none, little strong and very strong.
Akkim et al. 4789 Lytic enzymes assay Protease production was assayed using skim milk agar. Bacterial cells were spot inoculated and incubated for two days at 28°C. Proteolytic activities were identified by clear zone formation around the cell (Smibert and Krieg, 1994). Bacterial strains were screened for their chitinase activity on minimal salt (MS) chitin agar plates containing 0.1% (w/v) chitin with MS medium (0.5% yeast extract, 0.2% K 2HPO 4, 0.1% KH 2PO 4, 0.07% MgSO 4.7H 2O, 0.05% NaCl, 0.05% KCl, 0.01% CaCl 2, 1.3% bacto agar [pH 6.8]) and incubated at 30°C for seven days. The halo forming strains on the MS chitin plates were selected as chitin producers.β-1, 3-glucanase activity was estimated on plates containing minimal medium containing laminarin (Dunne et al., 1996) as sole substrates. Activity of β-1, 3- glucanase was determined by the ability of bacteria to grow on laminarin as the sole carbon source. HCN production Production of HCN was assessed on Kings’ B medium (KB) containing 4.4 g/L of glycine with indicator paper (Whatman no 1 soaked in 0.5% (w/v) picric acid and 2% (w/v) sodium carbonate) and plates incubated at 27°C for 48 to 72 h. Any positive response caused the indicator paper to turn from yellow to cream, light brown, dark brown and brick (Alstrom and Burns, 1989). Effects of volatile metabolites The effect of volatile metabolites of fluorescen pseudomonads on growth of M. phaseolina was studied by a paired Petri dish technique (Gagne et al., 1991). A fresh 24 h old bacterial culture was uniformly inoculated on King’s B agar plates. In another set PDA plates were inoculated at the centre with a six mm fungal disc from a three days old culture. The PDA plate with the fungus (downward facing) was then paired with the Petri dish containing the bacteria (upward facing) and sealed with parafilm. Uninoculated plates paired with PDA plates inoculated with fungus only served as control. The paired plates were incubated at 28°C and fungal colony diameters were measured six days after incubation. Detection of fluorescein and pyocyanin Pseudomonas agar F (Casein enzymic hydrolysate, 10 g; protease peptone, 10 g; K 2HPO 4, 1.5 g; MgSO 4, 1.5 g; distilled water, 1l) favours the formation of fluorescein whereas Pseudomonas agar P (Peptone, 20 g; MgCl 2, 1.4 g; K 2SO 4, 10 g; Agar, 15 g; Distilled water, 1l) stimulates the pyocyanin production and reduces fluorescein formation (King et al., 1954). All the six isolates of fluorescent pseudomonads were tested for production of fluorescein and pyocyanin. Pot culture experiment A pot culture experiment was laid out in completely randomized design to test the efficacy of Pseudomonas strains, that is, Pf1 and FP7 and pre mixture fungicide (Carbendazim + Mancozeb) in controlling the root rot disease, changes in growth promotion and yield parameters of Coleus. Potting medium (red soil: Cow dung: manure at 1:1:1 w/w/w) was autoclaved for 1 h for two consecutive days. The virulent strain of M. phaseolina was mass multiplied in sand maize medium (sand and maize powder at the ratio of 19:1) and incorporated in the soil at the rate of 10 g per kg of soil. The coleus cuttings were planted in the inoculated pots. The different treatments are: T1: Pf1 stem cuttings dip alone at 0.2%, T2: FP 7 stem cuttings dip alone at 0.2%, T3: Pre mixture fungicide (Carbendazim + Mancozeb) stem cuttings dip alone at 0.1%, T4: Pf1 stem cuttings dip + Soil drenching at 0.2% each, T5: FP 7 stem cuttings dip + Soil drenching at 0.2% each, T6: Pre mixture fungicide (Carbendazim + Mancozeb) stem cuttings dip + soil drenching at 0.1% each, T7: Pf1 soil drenching alone at 0.2%, T8: FP 7 soil drenching alone at 0.2%, T9: Pre mixture fungicide (Carbendazim + Mancozeb) soil drenching alone at 0.1%, T10: Inoculated control. Treatments were inoculated at monthly interval. Three replications (Three pots per replication) were maintained and pots were arranged in a randomized manner. The root rot incidence of M. phaseolina was recorded and expressed as percentage of disease incidence. Plant height, number of branches and bacterial population were recorded at monthly interval until harvesting. Harvesting was done after 140 days of transplanting. After harvesting different parameters viz., tuber length, tuber weight/plant and number of tubers/plant were recorded. Assay of defence enzymes Plant leaf tissues were collected at different time intervals (0, 24, 48, 72, 96, 164 h after pathogen inoculation). Four plants were sampled from each replication of the treatment separately and were maintained for biochemical analysis. Leaf samples were homogenised with liquid nitrogen in a pre-chilled mortar and pestle. One gram of leaf sample was homogenised with 2 ml of 0.1 M sodium phosphate buffer (pH 7.0) at 4°C. The homogenate was centrifuged for 20 min at 10,000 rpm. The supernatant was used as a crude enzyme extract for assaying peroxidase (PO) (Hammerschmidt and Kuc, 1982) and polyphenol oxidase (PPO) (Mayer et al., 1965). Native polyacrylamide gel electrophoresis analysis The isoform profiles of PO and PPO were studied by discontinuous native polyacrylamide gel electrophoresis (PAGE). The protein extract was prepared by homogenising 1 g of leaf sample in 2 ml of 0.1 M sodium phosphate buffer pH 7.0 and centrifuged at 16,000 g for 20 min at 4°C. The protein content of the sample was determined (Bradford, 1976) and samples (50 µg protein) were loaded into 8% polyacrylamide gels (Sigma, USA). After electrophoresis, PO isoforms were visualised by soaking the gels in staining solution containing 0.05% benzidine (Sigma Aldrich, Mumbai, India) and 0.03% H 2O 2 in acetate buffer (20 mM, pH 4.2) (Nadolny and Sequeira, 1980). For assessing the PPO isoform profiles, the gels were equilibrated for 30 min in 0.1% p-phenylene diamine, followed by the addition of 10 mM catechol in the same buffer (Jayaraman et al., 1987). Field experiment Two field trials were conducted at Periyanayakapalem, Coimbatore (district) and Attur, Salem (district) Tamil Nadu to test the efficacy of Pseudomonas isolates (Pf1 and FP 7) and pre mixture fungicide (Carbendazim + Mancozeb) (Carbendazim + Mancozeb) in controlling the root rot disease, growth and yield parameters of coleus. A randomized block design was used in the experiment. Treatments, replications and observations taken were similar as described in pot culture experiment.
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